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ABSTRACT Objective: To describe a protocol of obtention of mesenchymal stem cells and to report their use as a biological adjuvant in three patients undergoing arthroscopic rotator cuff repair. Methods: Case series of patients who underwent arthroscopic repair of isolated full-thickness supraspinatus tear using mesenchymal stem cells obtained from the bone marrow as a biological adjuvant. All patients were operated on at the same institution, by a surgeon with 13 years of experience. The cells were applied at the end of the procedure, at the tendon-bone interface, at an approximate concentration of 2,000,000 mesenchymal cells/mm3 and a total volume of 5 ml. Results: All patients improved with the procedure, with one excellent and two good results. All cases overcame the minimally important clinical difference. All cases reached tendon healing, without partial or complete re-tears. We observed no complications. Conclusion: Arthroscopic rotator cuff repair with added mesenchymal cells obtained from bone marrow and submitted to a cell expansion process led to good functional results and healing in all cases in the sample, with no complications. Level of Evidence IV, Case Series.
RESUMO Objetivo: Descrever o protocolo de obtenção de células mesenquimais e relatar seu uso como adjuvante biológico em três pacientes submetidos ao reparo artroscópico do manguito rotador. Métodos: Série de casos de pacientes submetidos ao reparo artroscópico de rotura transfixante do músculo supraespinal utilizando como adjuvante biológico células mesenquimais obtidas da medula óssea. Todos ospacientes foram operados na mesma instituição por um cirurgião com 13 anos de experiência. As células foram aplicadas ao final do procedimento, na interface do tendão com o osso, na concentração aproximada de 2 milhões de células mesenquimais/mm3 e volume total de 5 ml. Resultados: Todos os pacientes melhoraram após o procedimento, havendo um resultado excelente e dois bons. Todos superaram a diferença clínica minimamente importante. Em todos os casos ocorreu cicatrização tendínea, sem a presença de rerroturas parciais ou completas. Não observamos complicações. Conclusão: O reparo do manguito rotador artroscópico com adição de células mesenquimais obtidas da medula óssea e submetidas a processo de expansão celular levou a bons resultados funcionais e cicatrização, sem complicações, em todos os casos da amostra. Nível de Evidência IV, Série de Casos.
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ObjectiveTo investigate the therapeutic effect of the frozen and fresh preparations of human umbilical cord mesenchymal stem cells （hUC-MSC） on a rat model of liver cirrhosis after transplantation via the portal vein or the caudal vein. MethodsA total of 70 specific pathogen-free healthy male Sprague-Dawley rats were randomly divided into normal group （13 rats fed with ordinary tap water and rat food） and liver cirrhosis model group （57 rats given subcutaneous multi-point injection of mixed carbon tetrachloride/olive oil solution）. At week 8， the growth of rats was observed for both groups， and 3 rats were selected from each group for histopathological examination to confirm the formation of liver cirrhosis. A total of 50 rats were selected from the liver cirrhosis model and were divided into model group， portal vein group+fresh cell preparation group， portal vein+frozen cell preparation group， caudal vein+fresh cell preparation group， and caudal vein+frozen cell preparation group using a random number table， with 10 rats in each group. Fresh or frozen hUC-MSC were transplanted via the portal vein or the caudal vein， and after 4 weeks of administration， the different groups were compared in terms of the changes in liver function parameters and liver fibrosis degree. Continuous data were expressed as mean±standard deviation， and the independent-samples t test was used for comparison between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsAt week 8 of modeling， the model group showed the formation of pseudolobules of different sizes in the liver and met the diagnostic criteria for liver cirrhosis， with significant increases in the levels of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， total bilirubin （TBil）， and alkaline phosphatase （ALP） compared with the normal group （all P<0.001）， suggesting that the rat model of liver cirrhosis was established successfully. There were significant differences in the levels of ALT， AST， TBil， and ALP between the five groups （F=232.00， 177.10， 112.30， 121.70， all P<0.001）. Further comparison between two groups showed that the model group had significantly higher levels of ALT， AST， TBil， and ALP than the normal group （all P<0.01）， and the portal vein group+fresh cell preparation group， the portal vein+frozen cell preparation group， the caudal vein+fresh cell preparation group， and the caudal vein+frozen cell preparation group had significantly lower levels of ALT， AST， TBil， and ALP than the model group （all P<0.01）. ConclusionThere are significant improvements in liver function and liver fibrosis degree in a rat model of liver cirrhosis at week 4 after the transplantation of hUC-MSC， and frozen or fresh cell preparation and different transplantation approaches have no significant influence on treatment outcome.
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Osteochondral lesions of the talus (OLT) frequently manifest following ankle joint trauma, causing ankle pain, swelling and impaired mobility, thereby significantly impeding daily activities of the patients. Presently, clinical treatment approaches encompass both conservative management and surgical intervention. Conservative management endeavors to alleviate symptoms, while patients experiencing persistent symptoms resort to surgical intervention. Commonly employed surgical treatments encompass bone marrow stimulation, autologous osteochondral transplantation, and allogeneic osteochondral transplantation. Bone marrow stimulation is employed as a therapeutic approach for the management of smaller OLT, demonstrating favorable short-term effectiveness; however, the long-term prognosis remains uncertain. Autologous osteochondral transplantation is a viable option for larger OLT lesions, albeit it carries the potential of complications at the donor site. Conversely, allogenic osteochondral transplantation exhibits a diminished success rate. In recent times, the utilization of cell transplantation techniques has garnered escalating interest in the treatment of OLT due to their capacity to regenerate cartilage resembling hyaline and their diverse range of cellular origins. The authors reviewed the progress of cell transplantation in the treatment of OLT, providing a reference for the clinical treatment.
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Abstract Objective To evaluate in vitro the viability of mesenchymal stem cells derived from adipose tissue (AD-MSCs) in different commercial solutions of hyaluronic acid (HA) before and after being sowed in collagen I/III membrane. Methods In the first stage, the interaction between AD-MSCs was analyzed with seven different commercial products of HA, phosphate buffered saline (PBS), and bovine fetal serum (BFS), performed by counting living and dead cells after 24, 48 and 72 hours. Five products with a higher number of living cells were selected and the interaction between HA with AD-MSCs and type I/III collagen membrane was evaluated by counting living and dead cells in the same time interval (24, 48 and 72 hours). Results In both situations analyzed (HA + AD-MSCs and HA + AD-MSCs + membrane), BFS presented the highest percentage of living cells after 24, 48 and 72 hours, a result higher than that of HA. Conclusion The association of HA with AD-MSCs, with or without membrane, showed no superiority in cell viability when compared with BFS.
Resumo Objetivo Avaliar in vitro a viabilidade das células-tronco mesenquimais derivadas do tecido adiposo (AD-CTMs) em diferentes soluções comerciais de ácido hialurônico (AH) antes e após serem semeadas em membrana de colágeno I/III. Métodos Na primeira etapa, analisou-se a interação entre AD-CTMs com sete diferentes produtos comerciais de AH, salina tamponada com fosfato (PBS, na sigla em inglês) e soro fetal bovino (SFB), realizada pela contagem das células vivas e mortas após 24, 48 e 72 horas. Foram selecionados cinco produtos com maior número de células vivas e avaliou-se a interação entre o AH com AD-CTMs e a membrana de colágeno tipo I/III pela contagem de células vivas e mortas no mesmo intervalo de tempo (24, 48 e 72 horas). Resultados Em ambas as situações analisadas (AH + AD-CTM e AH + AD-CTM + membrana), o SFB apresentou a maior porcentagem de células vivas após 24, 48 e 72 horas, resultado superior ao do AH. Conclusão A associação do AH com as AD-CTMs, com ou sem a membrana, não demonstrou superioridade na viabilidade celular quando comparado com SFB.
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In Vitro Techniques , Cartilage, Articular , Collagen Type I , Mesenchymal Stem Cell Transplantation , Hyaluronic AcidABSTRACT
Objective:To evaluate the safety and validity of enriched autologous bone marrow mesenchymal stem cells (BMSCs) and annular suture for repairing defect after lumbar discectomy.Methods:Enrichment of autologous BMSCs: autologous bone marrow blood was collected from 5 patients undergoing lumbar surgery, and nucleated cells were enriched on gelatin sponge particles by selective cell retention technique. From October 2016 to March 2019, 109 patients with lumbar disc herniation underwent discectomy with mobile microendoscopic discectomy technique, including 61 males and 48 females, aged 24-59 years. Discectomy group: 26 cases received simple discectomy. Suture group: 39 cases received annular suture after discectomy. BMSCs+suture group: 44 cases received intradisc transplantation of gelatin sponge particles enriched with autologous BMSCs and annular suture after discectomy. The perioperative conditions were recorded, with visual analogue scale (VAS), Oswestry dysfunction index (ODI), Pfirrmann grade of disc degeneration, disc height and degree of herniationevaluated after operation.Results:In enrichment test with flow cytometry, the enrichment multiple of nucleated cells and target cells was 6.4±0.9 and 4.2±0.6 respectively, and BMSCs grew well in vitro. The operation time was 35-55 mins. 7 cases in the suture group were transferred to the discectomy group and 10 cases in the BMSCs+suture group were transferred to BMSCs group due to unsuccessful suture. There were no significant differences in VAS, ODI, Pfirrmann grade of disc degeneration, disc height and degree of herniation among the groups. There was no significant difference in intraoperative bleeding, postoperative drainage and length of hospital stay. The incision was healed without redness and swelling. 18 patients were followed up for 6 months, and 91 cases were followed up for 1-3 years (25.0±5.6 months). There was no interbody fusion, heterotopic ossification or infection during follow-up. VAS and ODI decreased significantly after operation in all patients. At final follow-up, the VAS improvement rate of BMSCs+suture group (81.7%±7.9%) was higher than discectomy group (73.0%±8.9%), suture group (74.0%±6.9%) and BMSCs group (75.3%±8.4%); the ODI improvement rate of BMSCs+suture group (91.9%±8.8%) was higher than discectomy group (86.2%±8.1%) and suture group (86.4%±5.5%). According to MRI, the Pfirrmann grade of disc increased 0.7 in discectomy group, 0.6 in suture group, while it did not increased significantly in BMSCs+suture group and BMSCs group, and the progress of Pfirrmann grade in BMSCs+suture group and BMSCs group were lighter than discectomy group and suture group.The disc height decreased in each group, the loss rate of disc height in BMSCs+suture group (17.2%±4.3%) was less than discectomy group (29.3%± 6.3%) and suture group (20.6%±5.7%); and suture group was less than discectomy group. The degree of herniation was reduced by more than 50% in all groups, while 1 case in discectomy group had herniation without clinical symptoms.Conclusion:Autologous BMSCs and annulus suture are safe and effective in repairing the defect after lumbar discectomy, which may help to slow down the degeneration of intervertebral disc.
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ABSTRACT Background: Crohn's disease is a pathological condition that has different options of treatment, but there are patients who need other therapeutic approach, such as the use of adipose-derived mesenchymal stem cells. Aim: Systematic literature review to determine the different ways of adipose-derived mesenchymal stem cells administration in humans with luminal refractory and perianal fistulizing Crohn's disease. Methods: It was conducted a search for articles (from 2008 to 2018) on PubMed and ScienceDirect databases using the keywords Crohn's disease, fistulizing Crohn's disease, luminal Crohn's disease and transplantation of mesenchymal stem cells or mesenchymal stem cells or stromal cells. Thirteen publications were selected for analysis. Results: Only one study referred to the luminal Crohn´s disease. The number of cells administered was variable, occurring mainly through subcutaneous adipose tissue by liposuction. It could be highlighted the autologous transplant with exclusive infusion of mesenchymal stem cells. The procedures involved in pre-transplant were mainly curettage, setons placement and stitching with absorbable suture, and conducting tests and drug treatment for luminal Crohn´s disease. During transplant, the injection of mesenchymal stem cells across the fistula path during the transplant was mainly on the intestinal tract wall. Conclusion: Although the use of mesenchymal stem cells is promising, the transplant on the luminal region should be more investigated. The injection of mesenchymal stem cells, exclusively, is more explored when compared to treatment with other products. The preparation of the fistulizing tract and the location of cell transplantation involve standardized health care in most studies.
RESUMO Racional: Há diferentes opções de tratamento para a doença de Crohn, porém, em alguns casos, há a necessidade de outras abordagens terapêuticas, como o uso de células-tronco mesenquimais derivadas do tecido adiposo. Objetivo: Revisar sistematicamente a literatura para determinar as diferentes formas de administração das células-tronco mesenquimais derivadas do tecido adiposo em seres humanos com doença de Crohn refratária luminal e fistulizante perianal. Método: Buscaram-se artigos publicados entre 2008 e 2018 nas bases de dados PubMed e ScienceDirect, pelos descritores: Crohn's disease, fistulizing Crohns disease, luminal Crohns disease e transplantation of mesenchymal stem cells ou mesenchymal stem cell ou stromal cells. Treze artigos foram selecionados. Resultados: Somente um trabalho se referiu à doença luminal. A quantidade de células administradas foi variável, obtendo-se principalmente do tecido adiposo subcutâneo por lipoaspiração. Destacou-se o transplante autólogo com a infusão exclusiva de células-tronco mesenquimais. Os procedimentos realizados no pré-transplante foram principalmente o de curetagem, colocação de setons e suturas com fio absorvível, e de exames e tratamento medicamentoso para a doença luminal. No transplante, ocorreu a injeção das células por todo o trajeto fistuloso, principalmente nas paredes do trato. Conclusão: Embora o uso de células-tronco mesenquimais seja promissor, o transplante na região luminal deve ser mais investigado. A injeção exclusiva de células-tronco mesenquimais é mais explorada quando comparada ao tratamento conjunto com outros produtos. A forma de preparo do trato fistuloso e o local de transplante envolvem cuidados médicos padronizados na maioria dos estudos.
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Humans , Crohn Disease/therapy , Adipose Tissue/cytology , Rectal Fistula/therapy , Mesenchymal Stem Cell Transplantation/methods , Crohn Disease/complications , Adipose Tissue/transplantation , Rectal Fistula/etiologyABSTRACT
To investigate the effects of adipose-derived mesenchymal stem cells (AMSCs) from type 2 diabetes mellitus patients on wound healing of pressure ulcers in mice.Methods@#(1) In September 2016, the subcutaneous adipose tissue of a 60-year-old woman with type 2 diabetes mellitus was harvested, and then AMSCs were extracted by collagenase digestion and cultured. The third passage of cells were used for subsequent experiments. The morphology of cells was observed, and their osteogenic, chondrogenic, and adipogenic differentiation abilities were identified. The expressions of cell surface markers CD90, CD105, CD73, and CD34 were detected by flow cytometer (n=3). (2) Sixteen female C57BL/6 wild-type mice aged 6-8 weeks were selected, and one pressure ulcer wound was created on each side of the spine of each mouse by pressing the skin with two magnets. The two wounds of each mouse were paired and divided into diabetic AMSCs group and negative control group, injected with 100 μL phosphate buffer solution (PBS) containing green fluorescent protein-labeled AMSCs (1×106 cells) and 100 μL PBS, respectively. The wound healing status of the two groups within post injection day (PID) 21 was observed, and their wound healing rates on PID 5, 13, and 17 were calculated. Three mice were sacrificed on PID 11 and 21, respectively, and tissue of three wounds was harvested from each group. The skin structure was observed by hematoxylin-eosin staining, the collagen deposition was evaluated by Masson staining, and the positive expression of CD31, i. e., the number of new blood vessels was counted by immunohistochemistry. Wound tissue samples of two groups prepared on PID 21 as above-mentioned were harvested, and the positive cell rate of S100, representing the regeneration of Schwann cells, was detected by immunohistochemistry. Wound tissue samples of diabetic AMSCs group prepared on PID 11 as above-mentioned were harvested, and the colonization of AMSCs was observed by fluorescence tracer method. Data were processed with paired t test and Bonferroni correction.@*Results@#(1) The third passage of cells isolated and cultured from the subcutaneous adipose tissue of a type 2 diabetes mellitus patient grew adherently to the wall in a long spindle and vortex-like manner. After induction, the cells showed osteogenic, chondrogenic, and lipogenic differentiation abilities. The positive expression rates of CD90, CD105, and CD73 on the cell surface were higher than 90.00%, and the expression rate of CD34 was 0.46%. The cells were identified as AMSCs. (2) The mice wounds of diabetic AMSCs group healed quickly, and all the wounds healed completely on PID 17, while the mice wounds in negative control group were not completely closed at this time, and there was still scab on the surface. On PID 5, 13, and 17, the healing rates of mice wounds of diabetic AMSCs group were (35.6±6.5)%, (87.1±2.5)%, and 100.0%, respectively, significantly higher than (19.8±7.2)%, (66.2±5.2)%, and (86.9±5.3)% of negative control group (t=6.49, 14.31, 9.73, P<0.05). Compared with that of negative control group, the inflammatory cell infiltration was reduced in mice wounds tissue of diabetic AMSCs group on PID 11, and thicker epidermis and dermis as well as regenerated skin appendages were observed on PID 21. On PID 11 and 21, the collagen percentages of mice wounds tissue in diabetic AMSCs group was (48.3±1.3)% and (54.1±1.7)%, respectively, significantly higher than (41.4±1.7)% and (50.3±1.2)% of negative control group (t=6.98, 3.99, P<0.01). On PID 11 and 21, the numbers of new blood vessels in mice wounds tissue of diabetic AMSCs group were 17.2±1.3 and 18.0±2.1, respectively, significantly more than 8.0±1.4 and 14.0±1.5 of negative control group (t=10.69, 3.38, P<0.01). On PID 21, the S100 positive cell percentage in mice wounds tissue of diabetic AMSCs group was (1.76±0.12)%, significantly higher than (0.55±0.03)% of negative control group (t=21.68, P<0.001). On PID 11, the colonization of AMSCs in mice wounds tissue of diabetic AMSCs group was observed.@*Conclusions@#Transplantation of AMSCs from type 2 diabetic mellitus patients can accelerate wound healing of pressure ulcers in mice by promoting angiogenesis, collagen deposition, and Schwann cell regeneration.
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Objective To investigate the effects and the mechanism of thrombospondin 4 (Thbs4) gene-edited bone marrow mesenchymal stem cells (BMSCs) transplantation on vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in diabetic rats with hind limb ischemia. Methods Thirty Sprague-Dawley rats were randomly divided into the model group, BMSCs treatment group and Thbs4-BMSCs treatment group on average. After constructing the typeⅡdiabetic rat model with hind limb ischemia, 100μl normal saline, BMSCs suspension and Thbs4-BMSCs suspension (cell number: 2×106) were locally injected into the ischemic injury area of rats for the model group, BMSCs group and Thbs4-BMSCs group, respectively. The rats were sacrificed on the 14th day after stem cell transplantation, and the muscle tissues near the ischemic area were collected. The relative expression of VEGF and p-Smad2/3 protein was detected by Western Blot. The Ang-1 protein expression was detected by immunofluorescence staining. The levels of related genes were detected by qRT-PCR, and the von Willebrand Factor (vWF) protein expression was detected by immunohistochemistry staining. Results The relative expression levels of VEGF, Ang-1 and vWF protein in the Thbs4-BMSCs group were significantly higher than those in the model group and BMSCs group (VEGF protein:P<0.01 and P<0.05). The mRNA expression of VEGF and Ang-1 were significantly up-regulated, the differences were statistically significant(VEGF mRNA:all P<0.01;Ang-1:P<0.01 and P<0.05). The expression of p-Smad2/3 protein in the Thbs4-BMSCs group was significantly higher than that in the model group and the BMSCs treatment group (all P<0.01). The expression of p-Smad2/3 protein was significantly decreased after the addition of p-Smad2/3 pathway inhibitor, the differences were statistically significant (P<0.05). Conclusions Thbs4-BMSCs transplantation can effectively promote angiogenesis in diabetic rats with hind limb ischemia, and the effect of angiogenesis may be related to the activation of Smad2/3 signaling pathway.
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BACKGROUND: Type l diabetes mellitus is a T cell-mediated autoimmune disease resulting in pancreatic islet cell damage. In this study, immunotherapy was used to deal with type l diabetes mellitus and stem cell transplantation was used to repair damaged islet p cells, attempting to explore a new treatment for type l diabetes mellitus. OBJECTIVE: To observe the efficacy of human umbilical cord mesenchymal stem cells combined with immunotherapy for the treatment of type l diabetic mice. METHODS: Fifty BALB/c Foxp3-DTR-EGFP positive mice were selected, six of which were randomly selected as normal control group and the remaining of which were intraperitoneally injected with streptozotocin and diphtheria toxin to prepare an animal model of type l diabetes mellitus. After successful modeling, randomization was performed in model mice and there were four groups: model group (normal saline), immunotherapy group (subcutaneous injection of dexamethasone (10 μg) and insulin (10 μg) mixture), cell transplantation group (injection of human umbilical cord mesenchymal stem cells (1 X106 cells per mouse) through the tail vein, and combined treatment group (the combination of immunotherapy and cell transplantation as described above). At 4 weeks after treatment, changes in blood glucose, C-peptide, body mass, pancreatic histopathology and insulin-positive area were observed in each group. RESULTS AND CONCLUSION: (1) Compared with the normal control group, the blood glucose level of the model group increased (P < 0.01) the C peptide level and body mass decreased (P < 0.01), and the islet was severely atrophied, with decreased number of islet cells and reduced insulin-positive area. (2) Compared with the model group, the blood glucose level of the immunotherapy group decreased (P > 0.05), the C-peptide level and body mass did not change significantly (P > 0.05), the islet cells increased in number, and the insulin-positive area increased. (3) Compared with the model group, the blood glucose level of the cell transplantation group and the combined treatment group decreased (P > 0.05), the C peptide level and body mass increased (P < 0.05), the islet cells increased in number, and the insulin-positive area increased. These findings reveal that either human umbilical cord mesenchymal stem cells or immunotherapy can improve the islet function of type l diabetic mice, and the combination treatment has better outcomes.
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Objective To investigate the effects of umbilical cord mesenchymal stem cells (UCMSCs) on immune microenvironment and angiogenesis in patients with traumatic brain injury. Methods Cerebrospinal fluid (CSF) samples were divided into 4 groups, including normal group (n=6), traumatic brain injury group (n=6), traumatic brain injury+UCMSCs treatment group ( n=6 ) and craniocerebral trauma + conventional treatment group ( n=6 ) . The CSF samples were detected by liquid chromatography-mass spectrometry , and data were collected by data independent acquisition (DIA) technology. The differential proteins were screened by bioinformatics processing, and analyzed by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Results A total of 688 proteins were screened in CSF samples and reliably quantified. There were 38 differential proteins in the CSF of patients with traumatic brain injury after treatment with UCMSCs, including 20 up-regulated proteins and 18 down-regulated proteins. The results of GO analysis and KEGG analysis showed that the differential proteins were mainly immunoregulatory function-related proteins, angiogenesis-related proteins, and various connexins. Conclusions The main possible mechanism of UCMSCs in the treatment of traumatic brain injury is to regulate the stability of the immune microenvironment and to promote the regeneration and reconstruction of damaged brain tissue.
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Objective To evaluate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on transforming growth factor-β1 (TGF-β1)/Smad signaling pathway during acute lung injury (ALI) in rats.Methods Healthy clean-grade adult male Sprague-Dawley rats,aged 4-5 weeks,weighing 100-120 g,were selected,and BMSCs were prepared and cultured in vitro.Eighty-four healthy clean-grade adult male Sprague-Dawley rats,aged 4 weeks,weighing 170-190 g,were selected and divided into 4 groups (n=21 each) using a random number table method:control group (group C),group ALI,ALI plus BMSCs group (group ALI + BMSCs),and ALI plus phosphate buffer solution (PBS)group (group ALI+PBS).ALI was induced by intraperitoneally injecting 5 mg/kg lipopolysaccharide 0.5 ml in anesthetized rats.In group ALI+BMSCs,1×104 cells/ml BMSCs 0.5 ml (in PBS) was injected via the tail vein after intraperitoneal injection of lipopolysaccharide.PBS 0.5 ml was injected via the tail vein after intraperitoneal injection of lipopolysaccharide in group ALI+PBS.Arterial blood samples were collected at 6,24 and 48 h after injecting BMSCs for blood gas analysis and for determination of wet to dry weight ratio (W/D ratio),pathological changes (using haematoxylin and eosin staining),and expression of TGF-β1,Smad2 and Smad3 in lung tissues (by Western blot).Results Compared with group C,PaO2 was significantly decreased,PaCO2 and W/D ratio were increased,the expression of TGF-β1,Smad2 and Smad3 in lung tissues was up-regulated (P＜0.05),and the pathological changes of lung tissues were accentuated in ALI,ALI+BMSCs and ALI+PBS groups.Compared with group ALI,PaO2 was significantly increased,PaCO2and W/D ratio were decreased,the expression of TGF-β1,Smad2 and Smad3 in lung tissues was down-regulated (P＜0.05),and the pathological changes of lung tissues were significantly reduced in group ALI+BMSCs.Conclusion The mechanism by which BMSCs mitigates ALI may be associated with inhibiting TGF-β1/Smad signaling pathway in rats.
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BACKGROUND: Mesenchymal stem cells can protect and repair the liver of rats with liver failure, but the mechanisms are not completely clear. OBJECTIVE: To explore the protective effects and related mechanisms of intravenous injection of human adipose-derived mesenchymal stem cells on acute liver failure in rats. METHODS: Thirty-six Sprague-Dawley rats (provided by Qingdao Daren Fucheng Animal Husbandry Co., Ltd. in China) were randomly divided into control group, model group and transplantation group. Animal models of acute liver failure were established by intraperitoneal injection of D-galactosamine in the model group and the transplantation group. One day after modeling, the rats in the transplantation group were injected with human adipose-derived mesenchymal stem cell suspension, and those in the model group were injected with the same amount of saline. After 1 and 3 days of cell transplantation, the serum levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin were measured. Three days after cell transplantation, the serum levels of tumor necrosis factor-α, interleukin-6 and interleukin-10 were detected, the pathological changes of the rat liver were observed by hematoxylin-eosin staining, and the activity of glycogen synthase kinase-3β protein in the liver tissue was detected by western blot. RESULTS AND CONCLUSION: Compared with the model group, there was a significant reduction in the serum levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, tumor necrosis factor-α, interleukin-6 and interleukin-10 in the transplantation group (P < 0.05). Inflammation and necrosis of liver tissues in the transplant group were alleviated compared with the model group. The activity of glycogen synthase kinase 3β in the liver tissue of the transplanted group was lower than that of the model group (P < 0.05). Overall, these results indicate that human adipose-derived mesenchymal stem cells can alleviate hepatic inflammation and pathological injury, and improve the liver function in rats with acute hepatic failure. Moreover, the mechanism may be related to the inhibition of glycogen synthase kinase 3β activity.
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BACKGROUND: Thin endometrial diseases are a challenge in clinical treatment at present. Scholars have found that bone marrow mesenchymal stem cells (BMSCs) transplantation has its unique curative effect and advantages, but few studies have been conducted on pathway or gene control. OBJECTIVE: To observe the effect of BMSCs transplantation in rats with thin endometrium based on the HOXA10 regulatory network. METHODS: Twenty-one adult female Sprague-Dawley rats of SPF grade (provided by the Animal Experimental Center, Guangzhou University of Chinese Medicine in China) were randomly divided into three groups (n=7/group): control group, model group, and BMSCs group. In the latter two groups, a thin endometrium model was prepared in each rat by filling the uterine cavity with 95％ ethanol. In the control group, normal saline was injected to fill the uterine cavity of rats. After extraction of ethanol or normal saline, the rats in the BMSCs group were injected intrauterinely with 1 mL of BMSCs suspension (1×1010 cells/L) , and those in the control and model groups were given the same volume of normal saline. After two estrous cycles, the uterus of each rat was removed. Hematoxylin-eosin staining was used to measure the thickness of the endometrium. Immunohistochemistry was used to detect the expression of vimentin, keratin, vascular endothelial growth factor, leukemia inhibitory factor and integrin αvβ3. qRT-PCR was used to detect the relative transcription of HOXA10 and miR-196 b. RESULTS AND CONCLUSION: (1) Compared with the control group, the endometrial thickness of the rats were significantly thinner in the model and BMSCs groups (P < 0.05) , while the endometrial thickness in the BMSCs group was thicker than that in the model group (P < 0.05). (2) The mean absorbance values of endometrial vimentin, keratin, vascular endothelial growth factor, leukemia inhibitory factor and integrin αvβ3 were highest in the control group, higher in the BMSCs group and lowest in the model group, and there were significant differences between groups (P < 0.05). (3) The relative transcript level of HOXA10 gene in the model and BMSCs group was significantly lower than that in the control group, while the relative transcript level of HOXA10 gene in the BMSCs group was significantly higher than that in the model group (P < 0.05). The relative transcript level of miR-196 b in the model and BMSCs groups was significantly higher than that in the control group (P < 0.05) , while the relative transcript level of miR-196 b in the BMSCs group was lower than that in the model group (P < 0.05). (4) HOXA10 was negatively correlated with miR-196 b gene, HOXA10 was positively correlated with the protein expression to different extents, and miR-196 b gene was negatively correlated with the protein expression to different extents. These findings suggest that BMSCs transplantation can improve the endometrial thickness and relevant protein levels of thin endometrium rats to some extent, which may be attributed to the negative regulation of HOXA10 gene by miR-196 b, and HOXA10 gene further promotes the expression of vascular endothelial growth factor, leukemia inhibitory factor and integrin αvβ3 proteins.
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Objective To evaluate the role of miR-146a in bone marrow mesenchymal stem cells ( BMSCs)-induced reduction of acute lung injury ( ALI ) in rats. Methods A total of 105 clean-grade healthy male Wistar rats, weighing 170-190 g, were divided into 5 groups ( n=21 each) using a random number table method: control group (group C), phosphate buffer solution group (group P), group ALI, BMSC group ( group B) and BMSC plus miR-146a inhibitor group ( group BM) . ALI was induced by intra-peritoneally injecting 5 mg∕kg lipopolysaccharide ( LPS) 0. 5 ml in anesthetized rats. Phosphate buffer solu-tion 0. 5 ml was injected via the tail vein in group P. In group B, 1×104 cells∕ml BMSC 0. 5 ml was injected via the tail vein after establishing the model. In group BM, miR-146a inhibitor 50 mg∕kg was injected via the tail vein after establishing the model, and 2 h later 1×104 cells∕ml BMSC 0. 5 ml was injected via the tail vein. Group C received no treatment. Blood samples were obtained from the abdominal aorta for blood gas a-nalysis at 6, 24 and 48 h after injection of BMSC ( T1-3 ) , the chest was immediately opened, and the lung tissues were obtained for microscopic examination of pathologic changes and for determination of the wet∕dry lung weight ratio ( W∕D ratio) , expression of IRAK-1, nuclear factor kappa B ( NF-κB) and interleukin-6 ( IL-6) ( by Western blot) and expression of miR-146a and IRAK-1 mRNA ( by quantitative real-time poly-merase chain reaction). Results Compared with group C, the pH value and PO2 were significantly de-creased, PCO2 and W∕D ratio were increased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was up-regulated at each time point ( P<0. 05) , and the pathological changes of lung tissues were aggrava-ted in ALI, B and BM groups. Compared with group P, the pH value and PO2 were significantly decreased, PCO2 and W∕D ratio were increased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was up-regulated at each time point (P<0. 05), and the pathological changes of lung tissues were aggravated in ALI, B and BM groups. Compared with group ALI, the pH value and PO2 were significantly increased, PCO2 and W∕D ratio were decreased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was down-regulated at each time point ( P<0. 05 ) , and the pathological changes of lung tissues were attenuated in group B. Compared with group B, the pH value and PO2 were significantly decreased, PCO2 and W∕D ratio were increased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was up-regulated at each time point ( P<0. 05) , and the pathological changes of lung tissues were aggravated in group BM. Conclusion miR-146a is involved in BMSCs-induced reduction of ALI in rats.
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ABSTRACT Objective: The main purpose of this study is to evaluate, in vitro, the cytotoxicity of different commercial brands of hyaluronic acids to be used as a vehicle for injection of human adipose-derived mesenchymal stem cells (AD-MSCs). Methods: AD-MSCs were divided into seven groups: one control group where AD-MSCs were cultivated with phosphate-buffered saline (PBS) and six other groups where AD-MSCs were cultivated with different commercial brands of hyaluronic acid. AD-MSC viability analysis was performed after 4, 24, and 48 h in contact with each treatment, using the trypan staining method on a Countess automated cell counter (Thermo Fisher Scientific). Results: The results clearly demonstrated a significant difference in cell viability when AD-MSCs were exposed to different hyaluronic acids when compared with the control group. Conclusion: These data suggest that hyaluronic acid can be used as a vehicle for injection of human AD-MSCs, but caution is needed to choose the best product, aiming at its future therapeutic application.
RESUMO Objetivo: Avaliar in vitro, de forma direta, a citotoxicidade de ácidos hialurônicos como veículo de injeção para linhagens de células-tronco mesenquimais (CTMs) obtidas de tecido adiposo humano. Métodos: As CTMs foram divididas em sete grupos, os quais foram expostos ao ácido hialurônico de seis marcas comerciais, além do contato com tampão fosfato-salino PBS (grupo controle). Após quatro, 24 e 48 horas, foi feita a análise da viabilidade celular através do contador Countess pelo método de coloração com Trypan Blue (Thermo Fisher Scientific). Resultados: Os resultados demonstraram uma diferença significativa na viabilidade celular quando essas linhagens de CTMs foram expostas aos diferentes ácidos hialurônicos em comparação com o grupo controle. Conclusão: Os dados sugerem que o ácido hialurônico pode ser usado como veículo de injeção para CTMs, porém é necessária cautela na escolha do melhor produto para aplicação terapêutica futura.
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Arthroscopy , Cartilage, Articular , Cartilage Diseases , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , KneeABSTRACT
Objective@#To explore the allogeneic mouse adipose-derived mesenchymal stem cell (ADSC)-microporous sheep acellular dermal matrix (ADM) on healing of wound with full-thickness skin defect in mouse and the related mechanism.@*Methods@#One Kunming mouse was sacrificed by cervical dislocation to collect adipose tissue from inguinal region. Mouse ADSCs were isolated from the adipose tissue and cultured in vitro. Cells of the third passage were identified by cell adipogenic and osteogenic differentiation. The expressions of CD73, CD90, CD105, and CD34 were analyzed by flow cytometry. After one sheep was sacrificed, microporous sheep ADM was prepared from sheep back using decellularization method and freezing-thawing method. A 12 mm diameter, round, full-thickness skin defect wound was made on the back of each one of 36 Kunming mice. The wounds were covered by microporous sheep ADM. The mice were divided into group ADSC and control (C) group with 18 mice in each group according to the random number table after surgery. A volume of 0.2 mL DMEM/F12 culture medium containing 1×106 ADSCs was injected between microporous sheep ADM and wound of mice in group ADSC. While 0.2 mL DMEM/F12 culture medium was injected between microporous sheep ADM and wound of mice in group C. On post surgery day (PSD) 12 and 17, wound healing rates of mice in the 2 groups were calculated. On PSD 7, 12, and 17, wound vascularization of mice in the 2 groups was observed under reverse irradiation of backlight. On PSD 7, 12, and 17, the wound granulation tissue of mice in group ADSC was observed by hematoxylin and eosin staining. On PSD 7, the thicknesses of granulation tissue of mice in the 2 groups was measured. On PSD 12 and 17, expressions of VEGF in wounds of mice in the 2 groups were detected by immunohistochemical method. The sample number was 6 in each group at each time point in the above experiments. Data were processed with t test and analysis of variance of factorial design.@*Results@#(1) After 7 days of adipogenic induction, lipid droplet was observed in cytoplasm using oil red O staining. After 21 days of osteogenic induction, black deposits of calcium salts were detected using silver nitrate staining. Expression rates of CD73, CD90, CD105, and CD34 in cells were 97.82%, 99.32%, 97.35%, and 5.88% respectively. The cells were identified as ADSCs. (2) The wound healing rates of mice in group ADSC on PSD 12 and 17 [(78±6)%, (98±3)%] were significantly higher than those in group C [(60±9)%, (90±4)%, t=4.26, 4.46, P<0.01]. (3) On PSD 7, no vessel obviously grew into the center of wounds of mice in the 2 groups, while the granulation tissue has covered the wounds of mice in group ADSC. On PSD 12, the vessels were more abundant in wounds of mice in group ADSC than those in group C. On PSD 17, big vessels crossing the whole wounds was observed in wounds of mice in group ADSC, while big vessels were observed without crossing the whole wounds in wounds of mice in group C. (4) The wounds were covered with thin granulation tissue on PSD 7, and the granulation tissue began to thicken on PSD 12 and were covered by epidermis on PSD 17 in wounds of mice in group ADSC. On PSD 7, the granulation tissue in wounds of mice in group ADSC [(0.62±0.05) mm] was significantly thicker than that in group C [ (0.31±0.04) mm, t=12.27, P<0.01]. (5) On PSD 12 and 17, expressions of VEGF in wounds of mice in group ADSC [(80.7±2.2), (0.98±0.03)/mm2] were significantly than those in group C [(59.5±2.4), (81.5±2.6)/mm2, t=15.95, 14.14, P<0.01].@*Conclusions@#Allogeneic mouse ADSC-microporous sheep ADM can accelerate angiogenesis and growth of granulation tissue, thus promoting wound healing, which may be due to the increase of expression of VEGF.
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Objective@#To investigate the effects of human adipose-derived mesenchymal stem cells (ADSCs) and platelet-rich plasma (PRP) on healing of wounds with full-thickness skin defects in mice.@*Methods@#ADSCs were isolated from the lumbar and abdominal fat donated voluntarily by a healthy woman undergoing liposuction in the Department of Plastic Surgery of Guangzhou General Hospital of Guangzhou Military Area Command, and the cells were cultured and identified. ADSCs of the second passage were used in the following experiments. The venous blood of the volunteer was taken, and PRP was obtained by secondary centrifugation. Thirty-six C57BL/6 mice were divided into simple injury group (n=12), simple ADSCs treatment group (n=12), and ADSCs+ PRP treatment group (n=12) according to the random number table. Each mouse was inflicted with a 1 cm×1 cm wound with full-thickness skin defect on the back. Immediately after injury, the wounds of mice in simple injury group were subcutaneously injected with 1 mL normal saline, the wounds of mice in simple ADSCs treatment group were subcutaneously injected with 1 mL phosphate buffer solution-blended ADSCs suspension (with concentration of 5×105 /mL, the same below), and the wounds of mice in ADSCs+ PRP treatment group were subcutaneously injected with 1 mL mixture of PRP and ADSCs (1∶2 volume ratio). Three mice in each group were taken on post injury day (PID) 3, 5, 7, and 14 to observe the gross condition of wound, and the wound healing rate was calculated. On PID 3, 5, and 7, the non-healing wound tissue and 0.5 cm normal skin tissue around the wound margin were taken after gross observation. The inflammation, re-epithelialization, and angiogenesis of tissue were observed by hematoxylin and eosin staining, and the re-epithelialization rate was calculated. The collagen synthesis of tissue was observed by masson staining. Immunohistochemistry was used to observe the expression of macrophages of tissue samples collected on PID 3 and 5. Data were processed with analysis of variance of factorial design and Least-Significant Difference test.@*Results@#(1) On PID 3, the wounds of mice in ADSCs+ PRP treatment group were with granulation tissue regeneration, redness, and swelling, and the wounds of mice in the other two groups were ruddy and with effusion. On PID 5, the wounds of mice in ADSCs+ PRP treatment group had less redness and swelling, which were dry with obvious scab, and wounds of mice in the other two groups were obviously red and swollen. On PID 7, scab formed basically on wounds of mice in the three groups. On PID 14, the wounds of mice in the three groups basically healed, and their crusts were off. On PID 3, 5, 7, and 14, the wound healing rates of mice in ADSCs+ PRP treatment group were obviously higher than those of the other two groups (P<0.05 or P<0.01). On PID 5 and 7, the wound healing rates of mice in simple ADSCs treatment group were obviously higher than those of simple injury group (P<0.01). (2) On PID 3, granulation tissue regeneration of wounds in ADSCs+ PRP treatment group was more than that in the other two groups. On PID 5, inflammatory reaction of wounds of mice was mild in ADSCs+ PRP treatment group, which was severe in the other two groups. On PID 7, the re-epithelialization process of wounds of mice was almost completed in ADSCs+ PRP treatment group, and the number of new vessels was more in ADSCs+ PRP treatment group than in the other two groups. The migration distance of regenerated epithelia around the wound edge in simple injury group and simple ADSCs treatment group was short. On PID 3, 5, and 7, the re-epithelialization rates of wounds of mice in ADSCs+ PRP treatment group were (37.6±4.5)%, (59.1±1.3)%, and (89.2±4.3)%, respectively, significantly higher than (25.7±1.5)%, (34.5±4.4)%, and (50.8±2.7)% in simple injury group and (29.1±0.8)%, (42.6±2.9)%, and (72.9±3.0)% in simple ADSCs treatment group (P<0.01). On PID 5 and 7, the re-epithelialization rates of wounds of mice in simple ADSCs treatment group were significantly higher than those in simple injury group (P<0.05 or P<0.01). (3) On PID 3 and 5, a quite large number of new collagen fibers appeared in granulation tissue of wounds of ADSCs+ PRP treatment group, while the collagen fibers in the other two groups were less. On PID 7, the granulation tissue of mice in ADSCs+ PRP treatment group decreased, and a large number of new collagen fibers appeared. The collagen fibers in wounds tissue of mice in simple ADSCs treatment group increased, while the collagen fibers deposited in wounds tissue of mice in simple injury group was still less. (4) On PID 3 and 5, the numbers of macrophages in wounds tissue of mice in simple ADSCs treatment group were 4.7±0.6 and 5.3±0.6 respectively, obviously lower than 6.3±0.6 and 7.7±0.6 in injury group (P<0.05 or P<0.01); the numbers of macrophages in wounds tissue of mice in ADSCs+ PRP treatment group were 3.0±1.1 and 2.7±0.5, significantly lower than those in the other two groups (P<0.05 or P<0.01).@*Conclusions@#Human PRP and ADSCs are involved in the early inflammation, metaphase of tissue proliferation, and re-epithelialization and shaping process of late stage of wounds with full-thickness skin defects in mice. The combination of ADSCs and PRP may be a comparatively good combination to improve the speed and quality of wound healing.
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Objective To evaluate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on mammalian target of rapamycin (mTOR) signaling pathways in lung tissues of rats with acute lung injury (ALI).Methods Healthy pathogen-free adult male Sprague-Dawley rats were selected,and the BMSCs were obtained and cultured in vitro.One hundred and five healthy clean adult male SpragueDawley rats,weighing 170-190 g,were divided into 5 groups (n=21 each) using a random number table:control group (group C),PBS group,group ALI,ALI plus BMSC group (group ALI+BMSCs),and ALI plus phosphate buffer solution (PBS) group (group ALI+PBS).Group C received no treatment.PBS 0.5 ml was injected via the tail vein in group PBS.Lipopolysaccharide (LPS,0.5 ml) 5 mg/kg was intraperitoneally injected to establish the model of ALI in group ALI.BMSCs (0.5 ml) 1×104 cells/ml were injected via the tail vein after intraperitoneal injection of LPS in group ALI+ BMSCs.PBS 0.5 ml was injected via the tail vein after intraperitoneal injection of LPS in group ALI+PBS.Arterial blood samples were collected for blood gas analysis at 6,24 and 48 h after injection of BMSCs.Lungs were then removed for determination of wet/dry weight ratio (W/D ratio) and expression of mTOR,nuclear factor kappa B (NF-κB) and tumor necrosis factor-alpha (TNF-o) in lung tissues (by Western blot) and for examination of the pathologic changes of lungs tissues (using haematoxylin and eosin staining).Results Compared with group C,pH value and PaO2 were significantly decreased,PaCO2 and W/D ratio were increased,and the expression of mTOR,NF-κB and TNF-α was up-regulated at each time point in ALI,ALI+BMSCs and ALI+PBS groups (P＜0.05).Compared with group ALI,pH value and PaO2 were significantly increased,PaCO2 and W/D ratio were decreased,the expression of mTOR,NF-κB and TNF-α was down-regulated at each time point (P＜0.05),and the pathologic changes of lungs tissues were significantly attenuated in group ALI+BMSCs.Conclusion The mechanism by which BMSCs reduce ALI may be associated with inhibiting mTOR signaling pathways in lung tissues of rats.
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Objective To investigate the effect of tissue engineered cartilage on the repair of artic ular cartilage defects in rabbits with calcium alginate gel (CAG) loaded transforming growth factor beta 3 (TGF-β3) and compounded with adipose mesenchymal stem cells (ADSCs).Methods The ADSCs were separated and cultured in subcutaneous fat of New Zealand white rabbits.The full-thickness articular cartilage defect model was made at the patellar groove by exposure of the femoral ankle joint.ADSCs were implanted into calcium alginate scaffolds loaded with TGF-β3 to construct tissue-engineered cartilage and transplanted into rabbit articular cartilage defects.The animals were divided into four groups:control group (injected with sterile isotonic saline),CAG group (injected with CAG),ADSCs + CAG group (injected with CAG loaded with ADSCs),TGF-β3 + CAG group (injected with CAG loaded with TGF-β3) and TGF-β3 + ADSCs + CAG group (injected with CAG loaded with TGF-β3 and ADSCs).At the end of 12 weeks,the repair of articular cartilage defects was observed by gross observation,hematoxylin-eosin (HE) staining,immunohistochemical staining of safranin-O and type Ⅱ] collagen,and the scoring method developed by In ternational Association of Cartilage Repair (ICRS) Histological score.Results The cartilage repair effect of TGF-β3 + ADSCs + CAG group was better than that of other groups,not only the neonatal tissue and the surrounding normal tissue closely,but also the secretion of extracellular matrix and normal tissue similar.The ICRS scores of each group were (7.06 ±+ 0.18) score,(7.15 + 0.23) score,(7.45 + 0.25) score,(7.47 + 0.24) score and (15.78 ±+ 0.24) score.That of TGF-β3 + ADSCs + CAG group was better than that of other groups,the difference was significant (P ＜ 0.05).Conclusions CAG loaded with TGF-β3 and combined with ADSCs has a good effect in repairing articular cartilage defects in rabbits,and is a structural repair.
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OBJECTIVES: Current treatments for osteoporosis were prevention of progression, yet it has been questionable in the stimulation of bone growth. The mesenchymal stem cells (MSCs) treatment for osteoporosis aims to induce differentiation of bone progenitor cells into bone-forming osteoblasts. We investigate whether human umbilical cord blood (hUCB)-MSCs transplantation may induce bone regeneration for osteoporotic rat model induced by ovariectomy. METHODS: The ovariectomized (OVX) group (n = 10) and OVX-MSCs group (n = 10) underwent bilateral ovariectomy to induce osteoporosis, while the Sham group (n = 10) underwent sham operation at aged 12 weeks. After a femoral defect was made at 9 months, Sham group and OVX group were injected with Hartmann solution, while the OVX-MSCs group was injected with Hartmann solution containing 1 × 107 hUCB-MSCs. The volume of regenerated bone was evaluated using micro-computed tomography at 4 and 8 weeks postoperation. RESULTS: At 4- and 8-week postoperation, the OVX group (5.0% ± 1.5%; 6.1% ± 0.7%) had a significantly lower regenerated bone volume than the Sham group (8.6% ± 1.3%; 12.0% ± 1.8%, P < 0.01), respectively. However, there was no significant difference between the OVX-MSCs and Sham groups. The OVX-MSCs group resulted in about 53% and 65% significantly higher new bone formation than the OVX group (7.7% ± 1.9%; 10.0% ± 2.9%, P < 0.05). CONCLUSIONS: hUCB-MSCs in bone defects may enhance bone regeneration in osteoporotic rat model similar to nonosteoporotic bone regeneration. hUCB-MSCs may be a promising alternative stem cell therapy for osteoporosis.